Sequences at the immediate 3′ terminus of several eukaryotic primary transcripts, synthesised just before the termination of transcription, are often lost during RNA processing. The rna82.1 mutation in Saccharomyces cerevisiae appears to result in a deficiency of the endonuclease that removes such sequences from certain yeast transcripts. Some small RNAs of rna82.1 cells are a few nucleotides longer than their counterparts in wild‐type S. cerevisiae. The 5S rRNAs made during very short pulse‐labellings of the mutant have, relative to the mature 121 nucleotide 5S RNA of wild‐type cells, an additional 7, 11 or 13 nucleotides at their 3′ terminus. These 5S forms reveal sites upon 5S genes where transcription probably terminates in vivo. The extra nucleotides upon 5S RNAs in rna82.1 cells are lost very slowly by sequential removal from the 3′ terminus. Through this 3′‐5′ exonuclease action the total 5S RNA of the mutant possesses several 3′‐terminal sequences yet is mostly only 0‐3 nucleotides longer than in wild‐type S. cerevisiae. Just one or two of these 3′‐terminal sequences serve as a substrate in vivo for a poly(A) polymerase since a small proportion of rna82.1 5S RNAs terminate in the sequence: CAAUCUUU(A)n.

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock, When S . cerevisiae growing exponentially on glucose media was shifted from 25 "C to 38 "C transient increases of 6 -7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rnal .I mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress.Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 "C to 38 "C) or severe (25 "C to 42 "C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heatshock and translated efficiently at supra-optimal temperatures.Upon exposure to a heat shock the cells of virtually all species display alterations in their physiology and protein synthesis that together constitute the 'heat-shock response' (reviewed in [l -31). While this response involves primarily an altered control over gene transcription, it also operates in eukaryotes partly through effects on the translocation of mRNAs from the nucleus or the stabilisation and selective translation of mRNAs in the cytoplasm [2, 31. The transcriptional changes involve stimulation of a small number of heat-shock protein (HSP) genes and the simultaneous repression of many, if not most, of the genes that were previously active. Of the 17 proteins whose synthesis is enhanced when Escherichia coli cells are heat-shocked, a few have been assigned either an enzymatic function (e.g. the 1ysU and lon gene products) or a role in the control of enzyme activities (e. g. rpoD which encodes the regulatory sigma subunit of E. coli RNA polymerase) [I]. All 17 genes are activated during heat-shock by the product of the htpR locus, a protein which displays both a striking structural similarity to sigma and the apparent ability to antagonize the activity of sigma [l].Correspondence to P. W. Piper, Department of Biochemistry, University College London, Gower Street, London, England WC1 E 6BTAbbreviations and nomenclature. The nomenclature used for yeast heat-shock ...

The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was inserted on a high copy plasmid into cells having a TRP1 gene disruption of the solitary chromosomal PGK locus. This enabled PGK transcripts of plasmid and chromosomal origin to be distinguished by virtue of their different sizes. Certain deletions lacking UASPGK displayed activities that were very low in unstressed cells, but which increased fifty to one-hundred fold after heat shock. With UASPGK present heat shock had only a relatively small or negligible effect on PGK mRNA levels. Heat shock activation was abolished when the -256 to -377 region with homology to the heat shock element consensus of eukaryotes was deleted in addition to UASPGK, but was unaffected by the deletion of regions further downstream containing TATA- and CAAT- sequence motifs. This is the first demonstration of a heat shock element, an activator site normally found upstream of eukaryotic heat shock protein genes, as a natural constituent of a high efficiency glycolytic promoter. It is proposed that PGK may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock.

Heat shock enhances the very high level of transcription of the phosphoglycerate kinase (PGK) gene in fermentative cultures of Saccharomyces cerevisiae. This response of PGK mRNA levels was not found on gluconeogenic carbon sources, and could be switched on or off subject to availability of fermentable carbon source. The addition of glucose to yeast growing on glycerol resulted in acquisition, within 30-60 min, of the ability to elevate PGK mRNA levels after heat shock. In addition, in aerobic cultures growing on glucose the exhaustion of the medium glucose coincided with a loss of the heat-shock effect on PGK mRNA and a switch-over to slower growth by aerobic respiration. Levels of hsp26 mRNA were analysed during these experiments. Contrasting with this requirement for fermentable catabolite for manifestation of a heat-shock response of PGK mRNA levels, the PGK enzyme was not synthesized at a greater level in heat-shocked fermentative than in gluconeogenic cultures. PGK is one of only a few proteins made efficiently after mild heat shock of yeast. Thus, heat-stress-induced elevation of PGK mRNA levels does not appreciably increase PGK synthesis during exposure to high temperatures and so its role may be to assist cells repressed in mitochondrial function during recovery following a heat shock.

In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII. Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat. Three variant 5S rRNA genes were isolated on the basis of such dissimilarity to rDNA repeat sequences. They display a remarkable conservation of their DNA in the vicinity of the 5S coding region, and are examples of a minor form of 5S rRNA coding sequence present in a small number of copies in the yeast genome. These variant sequences appear to be transcribed as efficiently as 5S rRNA genes of the rDNA repeat. In one of our isolates of the variant sequence a Ty transposable element is inserted 145bp upstream of the initiation point for 5S rRNA synthesis.